Phage display systems are regarded as a core technology platform for the construction and screening of polypeptide libraries, particularly antibody libraries. This is attributed to numerous practical considerations including, the availability of various genetic tools, the convenience of manipulation, and the high transformation efficiency of E coli cells. Today, naive antibody libraries displayed on phage are routinely used for antibody discovery, thereby obviating the need for animal immunizations and the use of traditional hybridoma technology. However, despite the successful use of phage display in antibody discovery and engineering protocols, there are a number of drawbacks associated with the expression and display of eukaryotic proteins in prokaryotic systems.
For example, some eukaryotic proteins cannot be functionally expressed in prokaryotic cells. In addition, prokaryotic host cells are typically not able to accomplish the full range of post-translational modifications that are characteristic of eukaryotic host cells. Some of the limitations associated with the use of a prokaryotic display system can be overcome by the use of a eukaryotic display system. For example, a unique advantage associated with the use of a yeast display system is attributed to the fact that yeast cells can be cultivated to high densities using relatively simple and inexpensive culture medium. Generally speaking, eukaryotic host cells can accommodate the display of relatively large proteins, and are capable of post translation modifications including complex glycosylation. In addition, because eukaryotic cells are larger in size than prokaryotic cells, the members of a display libraries can be efficiently screened for single cells expressing proteins with desired properties (i.e., binding specificities) by flow cytometry.
The display of heterologous protein on the cell surface of Saccharornyces cerevisiae was first described in 1993 using a cell wall protein-based fusion protein design in which alpha-galactosidase was fused to the C-terminal half of cell wall protein alpha-agglutinin AGA 1 (Schreuder M P et al, Yeast 9:399-409). Since then, numerous yeast display systems based on fusing a library of proteins of interest to cell wall proteins have been reported (Kondo M et al). Among all of the cell wall fusion protein-based display systems, the system created by Dane Wittrup based on a-agglutinin receptor has been widely used to display various proteins libraries including various formats of antibody libraries (U.S. Pat. No. 6,300,065, U.S. Pat. No. 6,423,538, U.S. Pat. No. 6,696,251, and U.S. Pat. No. 6,699,658).
Similarly, a number of approaches have been designed to achieve the display of proteins on the surface of mammalian cells using display vectors which comprise a membrane anchor proteins fused to the members of a protein library comprising a diverse repertoire of protein sequences. Typically, the anchor protein comprises a protein derived from the membrane domain of a cell surface receptor (Chestnut et al, 1996, J Immunological Methods; Ho et al, 2006, PNAS, 103:9637-9642), such as a GPI anchor sequences (U.S. Pat. No. 6,838,446), or a non-cleavable type 11 signal anchor sequence (U.S. Pat. No. 7,125,973). For example, the pDISPLAY vector (Invitrogen Life Technologies), is a commercially available vector which directs the cell surface display of proteins on mammalian cell utilizes the membrane domain of cell surface platelet derived growth factor receptor PDGFR. Proteins expressed from the pDISPLAY vector are anchored to the plasma membrane of the host cell and displayed on the extracellular side of the plasma membrane.
There are a number of drawbacks associated with the use of a cell surface display system based on fusing the protein library to a cell surface anchor protein. For example, because the proteins of interest are directly fused to the outer surface anchor protein, the protein of interest can only be expressed as a part of the membrane protein. In order to obtain soluble protein for evaluation in screening and/or functional assays, additional molecular cloning steps are required in order to transfer the coding sequences of interest to an expression vector which directs the expression of soluble protein. Use of a cell wall fusion protein-based design also eliminates the possibility of evaluating the functional properties of expressed proteins inside cellular organelles such as mitochondria, Golgi apparatus, endoplasmic reticulum etc.
Therefore, there is an unmet need for an alternative protein display system which facilitates the display of protein libraries on eukaryotic cell surfaces, using a vector design which can also be used to direct expression of library proteins as either soluble proteins or as intracellular proteins without any molecular manipulations to reengineer the display vector.